Cytotoxicity studies of a stilbene extract and its main components intended to be used as preservative in the wine industry

1 Cytotoxicity studies of a stilbene extract and its main components 1 intended to be used as preservative in the wine industry 2 Concepción Medrano-Padiala, María Puertoa*, María del Mar Merchán-Grageroa, F. 3 Javier Morenob, Tristan Richardc, Emma Cantos-Villard, Silvia Pichardoa 4 a Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García 5 González n°2, 41012 Seville. Spain. ([email protected]; mmerchan[email protected]s; 6 [email protected]) 7 b Area of Cellular Biology, Faculty of Biology, Universidad de Sevilla, Avda. Reina 8 Mercedes s/n, 41012 Seville, Spain ([email protected]) 9 c Faculté des Sciences Pharmaceutiques, Unité de Recherche OEnologie EA 4577, 10 USC 1366 INRA, Equipe Molécules d’Intérêt Biologique (Gesvab). Institut des 11 Sciences de la Vigne et du Vin, Université de Bordeaux, CS 50008 210, Chemin de 12 Leysotte, 33882 Villenave d’Ornon, France. ([email protected]) 13 d Instituto de Investigación y Formación Agraria y Pesquera (IFAPA). Centro Rancho 14 de la Merced. Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible. 15 Junta de Andalucía). Cañada de la Loba, km 2.1, 11471, Jerez de la Frontera, Spain. 16 ([email protected]) 17 18 *Corresponding author: 19 María Puerto 20 Area of Toxicology, Faculty of Pharmacy, University of Seville, Profesor García 21 González n°2, 41012 Seville. Spain. 22 E-mail address: [email protected] 23 Tel: +34 954 556762 24 Fax: +34 954 556422 25 Abbreviations 26 EC50: mean effective concentration; SO2: sulfur dioxide. IC: Combination Index 27 2 Abstract 28 The use of stilbenes has been proposed as an alternative to sulfur dioxide in wine. 29 Provided the feasibility from a technological approach, the cytotoxicity of an extract 30 from grapevine shoots containing a stilbene richness of 99% (ST-99 extract) was 31 assessed in the human cell lines HepG2 and Caco-2. In addition, the effects of the 32 main stilbenes found in ST-99, trans-resveratrol and trans-ε-viniferin were studied, as 33 well as its mixture. Similar cytotoxic effects were obtained in the exposures to trans-ε34 viniferin, ST-99 and the mixture; however, trans-resveratrol alone exerted less toxicity. 35 When HepG2 cells were exposed to trans-ε-viniferin, ST-99 and the mixture, the mean 36 effective concentration (EC50) were 28.28 ± 2.15, 31.91 ± 1.55 and 29.47 ± 3.54 µg/mL, 37 respectively. However, in the exposure to trans-resveratrol, the EC50 was higher 38 50µ/mL. The morphological study evidenced damage at ultrastructural level in HepG2 39 cells, highlighting the inhibition of cell proliferation and the induction of apoptosis. The 40 type of interaction produced by trans-ε-viniferin and trans-resveratrol mixtures was 41 assessed by an isobologram analysis using the CalcuSyn software, evidencing an 42 antagonist effect. These data comprise a starting point in the toxicological assessment; 43 further studies are needed in this field to assure the safety of the extract ST-99. 44 45 Keywords: toxicity; stilbene; wine; trans-resveratrol; trans-ε-viniferin; wine 46 47 3 1. Introduction 48 The most widely used preservative in wine industry is sulfur dioxide (SO2). However, 49 many side effects have been attributed to SO2 in sensitive human such as dermatitis, 50 urticarial, angioedema, diarrhea, abdominal pain, bronchoconstriction, and anaphylaxis 51 (Guerrero et al., 2015). In addition, the European Food Safety Authority (EFSA) has 52 recently recommended that the temporary group acceptable daily intake (ADI) for SO2 53 should be re-evaluated (EFSA, 2016a). The Panel also concluded that exposure 54 estimates to SO2 and sulfites were higher than the group ADI of 0.7 mg SO2 55 equivalent/kg bw per day for all population groups. Moreover, consumers demand 56 products containing natural ingredients, due to an increase in green awareness. 57 Considering all this background, the wine industry is searching for new alternatives to 58 SO2 trying to avoid synthetic preservatives. One of the most promising alternatives is 59 the use of phenolic compounds. Natural extracts rich in stilbenes have been assayed 60 for this purpose (Raposo et al., 2016). Grapevine shoot are particularly rich in 61 stilbenes, with trans-resveratrol and trans-ε-viniferin present in considerably high 62 amounts (Anastasiadi et al., 2012; Guerrero et al., 2016), showing high antioxidant and 63 antimicrobial properties (Biais et al., 2017; Müller et al., 2009; Ruiz-Moreno et al., 64 2015). In fact, previous studies carried out in our laboratory have checked the safety 65 and usefulness of a stilbene extract containing 45.4% of stilbenes (Medrano-Padial et 66 al., 2019). Further processes were able to obtain an extract with higher percentage of 67 stilbenes (99%) named ST-99, which has proved to have good properties to be used as 68 preservative in wines (data non-published). The next step is now to check its safety 69 regarding consumers. 70 The EFSA in the guidance on safety assessment of botanicals and botanical 71 preparations intended for use as ingredients in food supplements (EFSA, 2009) 72 advises that the studies to probe their safety should be carried out in accordance with 73 the principles of reduction, refinement and replacement. According to this guidance, the 74 4 first step should be in vitro studies. Moreover, in chemical mixture toxicology, it is 75 essential first to evaluate the toxicity profile with in vitro approaches that will provide 76 important information related to the mode of action (Hernandez et al., 2019). Therefore, 77 the present work aims to assess the cytotoxicity of the ST-99 extract in two human cell 78 lines, HepG2 (liver hepatocellular cells) and Caco-2 (epithelial colorectal 79 adenocarcinoma cells). The toxicity of synthetic trans-resveratrol is well characterized 80 as summarized in the scientific opinion about its safety to be used as a novel food 81 (EFSA, 2016b). The Panel concludes that synthetic trans-resveratrol does not raise 82 safety concerns at the intended intake level of 150 mg/day for adults. The toxicity of 83 trans-ε-viniferin has been faintly studied so far. Some of the authors studying the effect 84 of this compound reported no cytotoxic effect of trans-ε-viniferin at low concentrations. 85 Hence, Richard et al. (2011) evidenced that trans-ε-viniferin glucoside did not 86 significantly affect the viability in the neuronal cells PC12 exposed up to 10 µM. 87 Similarly, trans-ε-viniferin had no cytotoxic effect on neurons and astrocytes at 88 concentrations lower than 10 μM. Indeed, they found that trans-ε-viniferin preserved 89 neuronal integrity at 1µM (Vion et al., 2018). The cytotoxicity activity of trans-ε-viniferin 90 against mouse lymphoma cells (P-388) revealed a half-maximal inhibitory 91 concentration (IC50) of 18.1±0.7 µM (Muhtadi et al., 2006). Moreover, Nivelle et al. 92 (2018) demonstrates that trans-ε-viniferin present antitumoral activities on human 93 melanoma cells without toxicity on normal human dermal fibroblasts at concentrations 94 of 60-85 µM. 95 Consumers are exposed to stilbenes by ingestion of different foods that naturally 96 contain them, such as wines, berries, peanut and its derivatives, pistachio, nuts , dark 97 chocolate, and grapes and their derivatives and herbal plants contain (Baur and 98 Sinclair, 2006; Bavaresco et al., 2016; Guerrero et al., 2009; 2020). In this sense, the 99 amount of stilbenes daily intake is highly different around the world according to the 100 type of diet (El Khawand et al., 2018). However, due to this new application in the food 101 5 industry, the intake of these stilbenes may increase, and consequently an accurate 102 toxicological assessment is required. 103 Hence, the present work studied the cytotoxicity of the most relevant biologically active 104 constituents found in a grapevine shoot extract; trans-resveratrol and trans-ε-viniferin, 105 were also performed, alone and in a mixture of both with the same proportion found in 106 the extract (1:3.9). In addition, the effects of their combinations were studied by an 107 isobologram analysis in order to detect potential interactions between both stilbenes. 108 Moreover, the ultrastructural study performed in both cell lines exposed to the extract 109 and the mixture of stilbenes helped to clarify in the mechanism of action of the extract. 110 111 2. Materials and methods 112 2.1. Supplies and chemicals 113 Culture medium, fetal bovine serum and cell culture reagents were obtained from 114 Gibco (Biomol, Sevilla, Spain). Chemicals for the different assays were provided by 115 Sigma-Aldrich (Madrid, Spain), (Biotech Ibérica, Madrid, Spain) and VWR International 116 Eurolab (Barcelona, Spain). 117 Trans-resveratrol was provided by Sigma–Aldrich (≥99% pure as determined by 118 HPLC). Trans-ε-viniferin was obtained from grapevine stems harvested in Bordeaux 119 region (France) and were composed of a mixture of Merlot and Cabernet Sauvignon 120 varieties of Vitis vinifera. Trans-ε-viniferin (98%) was purified by preparative HPLC as 121 reported by Gabaston et al. (2018). 122 123 2.2. Grapevine-shoot extract preparation and test solutions 124 The protocol used to obtain the grapevine-shoot extract was reported in a previous 125 work (Gabaston et al., 2018). Dried and finely ground vineshoot of V. vinifera cv. were 126 extracted with acetone–water (6:4, v/v) at room temperature under agitation, twice for 127 12 h. After filtration, the solution was submitted to evaporation under reduced pressure 128 6 and lyophilisated. Finally, the extract was deposited on an Amberlite XAD-7 column 129 and washed with water. The column was then eluted with acetone. The solvent was 130 evaporated until dryness. The extract was first solved in Arizona K solvents and 131 filtrated. Furthermore, the extract was fractionated by centrifugal partition 132 chromatography (CPC) and analyzed by UHPLC-MS using the method developed by 133 Biais et al. (2017). The stilbene fraction enriched in trans-resveratrol and trans-ε134 viniferin was collected and named ST-99. The ST-99 extract contained at least 99% of 135 total stilbenes (w/w), being the main stilbenes found trans-ε-viniferin (70%) and trans136 resveratrol (18%). Other stilbenes found in a lower percentage are vitisin B (4%), w137 viniferin (4%), cis-ε-viniferin (1%), miyabenol C (1.5%), and cis-resveratrol (0.5%) 138 The range of the extract and trans-ε-viniferin concentrations for the cytotoxicity tests 139 was selected considering the concentration to be incorporated in wine (100 mg/L). 140 However, in the case of trans-resveratrol, the maximum concentration used was 50 141 µg/mL because it was the highest concentration showing adequate solubility and it is 142 within the concentration range of this compound that will reach the consumer. Serial 143 test solutions (0-100 µg/mL) were prepared from stock solution (1000 µg/mL) in 144 dimethylsulfoxide (DMSO), being the final concentration in DMSO below 0.5%. 145 146 2.3. Model systems 147 The Caco-2 cell line derived from a human colon carcinoma (HTB-37) and HepG2, a 148 human hepatocellular carcinoma epithelial cell line (HB-8065), were maintained at 149 37ºC in an atmosphere containing 5% CO2 at 95% relative humidity (CO2 incubator, 150 Nuaire®, Spain). Caco-2 cells were cultured in a medium consisting of Eagle’s medium 151 (EMEM) supplemented with 20% foetal bovine serum (FBS), 1% non-essential amino 152 acids, 50 g/ml gentamicin, 2 mM L-glutamine and 1 mM pyruvate. HepG2 cells were 153 cultured in monolayer in EMEM supplemented with 10% of FBS, 100 U/ml penicillin 154 7 and 2 mM L-glutamine. Cells were grown 80% confluent in 75-cm2 plastic flasks and 155 harvested 3 times weekly (1:2 split ratio) with 0.25% trypsin. 156 157 2.4. Cytotoxicity assays 158 For the cytotoxicity assays, both cell lines were seeded in 96-well culture plates. 159 HepG2 cells were plated at density of 5 x 104 cells/ well and Caco-2 cells at 7.5 × 105 160 cells/ well to perform the experiments. 161 A wide range of concentrations in medium was prepared from the initial solution of 100 162 µg/ml. Culture medium without the extract was used as a control group. A control of 163 solvent (0.5% of DMSO) was also included. The cytotoxicity assays were performed in 164 cells exposed for 24 h and 48 h to ST-99 extract, trans-resveratrol, trans-ε-viniferin and 165 the mixture of both stilbenes in the same ratio that they are found in the extract 166 (1:3.9).Neutral red uptake (NR) was measured as described in Borenfreund & Puerner 167 (1984). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4168 sulfophenyl)-2H-tetrazolium salt) reduction was evaluated according to Baltrop et al. 169 (1991). The protein content (PC) assay was performed according to the procedure 170 given by Bradford (1976). 171 172 2.5. Assessment of the effect of stilbenes combination by the isobolograms method 173 In order to assess the effect of the stilbene’s combination, cells were exposed to 174 different concentrations, which were selected from the cytotoxicity tests of single 175 stilbenes. The mean effective concentration (EC50) values obtained for the most 176 sensitive endpoint at 24 h were chosen as the highest exposure concentrations, along 177 with EC50/2 and EC50/4 fractions. Thus, cells were exposed for 24 h and 48 h to binary 178 pure stilbenes mixtures: EC50 trans-resveratrol + EC50 trans-ε-viniferin, EC50/2 trans179 resveratrol + EC50/2 trans-ε-viniferin and EC50/4 trans-resveratrol + EC50/4 trans-ε180 8 viniferin. Moreover, each concentration used in the combinations was evaluated alone. 181 All experiments were performed by triplicate. 182 The isobologram analysis was carried out as described in Tatay et al. (2014), with 183 modifications (Gutiérrez-Praena et al., 2019). 184 According to Chou and Talalay (1984) and Chou (2006), the isobologram analysis 185 involves plotting the concentration-effect curves for each compound and its 186 combinations in multiple diluted concentrations by using the median-effect equation. 187 fa/fu = (D/Dm)m 188 D is the concentration of the stilbene, Dm the median-effect dose, fa is the fraction 189 affected by D, fu is the unaffected fraction, and m is the coefficient signifying the shape 190 of the dose–effect relationship. The method considers both the potency (Dm) and the 191 shape (m). 192 This single-dose equation can be extended for a multiple combination of stilbenes as 193 follows: 194 [(fa)1,2 / (fu) 1,2] 1/m = D1 /(Dm)1 + D2/ (Dm)2 + (D)1 (D)2 / (Dm)1 (Dm)2 195 This method provides the combination index (CI), which is useful for the quantification 196 of synergism, additivity or antagonism of two compounds. 197 CI = D1 / (Dx) 1 + D2/ (DX) 2 198 Dx= Dm [fa/ (1-fa)] 1/m 199 CI= (D)1 / (Dm)1[fa/(1-fa)]1/m1 + (D)2/(Dm)2 [fa/(1-fa)]1/m2 200 (Dx) 1 and (Dx) 2 are for D1 and D2 alone, respectively, that present a % effect on a 201 system. When the CI < 1, this suggests synergism; when CI is =1, it indicates additivity; 202 and when CI is >1, it refers antagonism. The CI50, CI75 and CI90 are the CI values at 203 50%, 75% and 90% inhibition, respectively. These CI values were calculated by the 204 CalcuSyn software (version 2.1.) (Biosoft, Cambridge, UK, 1996–2007). The 205 parameters Dm, m, and r of the combinations are the antilog of x-intercept, the slope 206 9 and the linear correlation coefficient of the median-effect plot, respectively, and they 207 give information about the shape of the concentration–effect curve. 208 209 2.6. Morphological study under transmission electron microscope 210 Electron microscope observations were performed according to Gutiérrez-Praena et al. 211 (2019). Cultured cells were exposed to three different concentrations of the extract and 212 the mixture, the EC50 value and their fractions (EC50/2, EC50/4). HepG2 were exposed 213 to 31.91, 15.95, and 7.98 µg/ml for the extract; and 29.47, 14.73, and 7.37 µg/ml for the 214 mixture. 215 216 2.7. Calculations and statistical analysis 217 Data for the concentration-dependent cytotoxicity relationships of all experiments were 218 expressed as the arithmetic mean percentage ± standard deviation (SD) in relation to 219 control. Statistical analysis performed was the analysis of variance (ANOVA), and 220 further the Dunnett’s multiple comparison tests was used. The normality of the 221 distribution and the homogeneity of variances were confirmed using Kolmogorov and 222 Smirnov’s test, and Bartlett´s test, respectively. All the analysis was carried out using 223 GraphPad InStat software (GraphPad Software Inc., La Jolla, USA). Differences were 224 considered significant in respect to the control group at p < 0.01 (*), p < 0.05 (**) and at 225 p < 0.01 (***). EC50 values were achieved by linear regression in the concentration226 response curves. 227 228 3. Results 229 3.1. Cytotoxicity studies of ST-99, individual stilbenes and their mixture. 230 The EC50 values corresponding to the cytotoxicity assays of HepG2 and Caco-2 cells 231 exposed to ST-99 extract, individual stilbenes and their mixture are shown in table 1. In 232 the case of trans-resveratrol the EC50 values in both cells could not be calculated 233 16 In conclusion, our results indicate a significant decrease in the viability of the human 394 intestinal Caco-2 cells and liver HepG2 cells after exposure to ST-99 extract, trans-ε395 viniferin and its mixture with trans-resveratrol (1:3.9) in the cytotoxicity assays, while 396 trans-resveratrol presented the lower effect. In addition, the type of interaction of trans397 resveratrol and trans-ε-viniferin was stablished by the isbolograms method reporting an 398 antagonistic response. The ultra-structural alterations in HepG2 cells exposed to ST-99 399 extract and the mixture evidenced that the cytotoxicity previously observed was due to 400 a breakdown in the cell cycle by inhibiting cell proliferation and induction of apoptosis. 401 These findings are of great concern not only because they contribute to increase the 402 knowledge of these stilbenes but also because the ST-99 extract could be used as an 403 alternative to SO2 in winemaking. Considering the toxicity observed in the in vitro 404 assays performed, further studies are needed in order to assess the toxicity on human 405 and ensure its safety. 406 407 5. Acknowledgements 408 The authors thank the CITIUS Biology Service (University of Seville) for the technical 409 assistance offered. Moreover, we would like to thank Dr. Gutierrez-Praena for his 410 assistance in the isobologram study. In addition, we thank M-L. Iglesias and A. Palos411 Pinto for their technical assistance. 412 Funding: This work was supported by the Ministerio de Economía, Industria y 413 Competitividad and INIA for the financial support for this project (RTA2015-00005-C02414 02). Moreover, it was also supported by the Bordeaux Metabolome Facility and 415 MetaboHUB (ANR-11-INBS-0010 project). 416 417 17 6. References 418 Anastasiadi, M., Pratsinis, H., Kletsas, D., Skaltsounis, A.L., & Haroutounian, S.A. 419 (2012). Grape stem extracts Polyphenolic content and assessment of their in 420 vitro antioxidant properties. LWTFood Science and Technology, 48, 316-322. 421 https://doi.org/10.1016/j.lwt.2012.04.006 422 Balasubramani, S.P., Rahman, M.A., & Basha, S.M. (2019). Synergistic action of 423 stilbenes in muscadine grape berry extract shows better cytotoxic potential 424 against cancer cells than resveratrol alone. Biomedicines. 7(4), 96. 425 https://doi.org/10.3390/biomedicines7040096 426 Baltrop, J.A., Owen, T.C., & Cory A.H. 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(2012). ε-Viniferin Is More Effective Than Its Monomer Resveratrol 587 in Improving the Functions of Vascular Endothelial Cells and the Heart. 588 Bioscience, Biotechnology and Biochemistry, 76:5, 954-960, 589 https://doi.org/10.1271/bbb.110975 590 591 592 593 22 Figure legends 594 Figure 1. Reduction of tetrazolium salt (MTS), neutral rep uptake (NR) and total protein 595 content (TP) of HepG2 cells exposed for 24 h (A) and 48 h (B) to 0-100 µg/mL of the 596 stilbene extract ST-99, and exposed for 24 h (C) and 48 h (D) to 0-100 µg/mL of the 597 stilbene mixture. All values expressed as mean ± SD. Significant differences in respect 598 to the control from p<0.01 (**). 599 Figure 2. Reduction of tetrazolium salt (MTS), neutral rep uptake (NR) and total protein 600 content (TP) of HepG-2 cells exposed for 24 h (A) and 48 h (B) to 0-100 µg/mL of 601 trans-ε-viniferin, and exposed for 24 h (C) and 48 h (D) to 0-50 µg/mL of trans602 resveratrol. All values expressed as mean ± SD. Significant differences in respect to 603 the control from p<0.05 (*) and p<0.01 (**). 604 Figure 3. Reduction of tetrazolium salt (MTS), neutral rep uptake (NR) and total protein 605 content (TP) of Caco-2 cells exposed for 24 h (A) and 48 h (B) to 0-100 µg/mL of the 606 stilbene extract ST-99, and exposed for 24 h (C) and 48 h (D) to 0-100 µg/mL of the 607 stilbene mixture. All values expressed as mean ± SD. Significant differences in respect 608 to the control from p<0.01 (**). 609 Figure 4. Reduction of tetrazolium salt (MTS), neutral rep uptake (NR) and total protein 610 content (TP) of Caco-2 cells exposed for 24 h (A) and 48 h (B) to 0-100 µg/mL of trans611 ε-viniferin, and exposed for 24 h (C) and 48 h (D) to 0-50 µg/mL of trans-resveratrol. All 612 values expressed as mean ± SD. Significant differences in respect to the control from 613 p<0.05 (*) and p<0.01 (**). 614 Figure 5. Combination index (CI)/fraction affected (fa) curve in HepG2 cells exposed to 615 a binary mixture of trans-ε-viniferin and trans-resveratrol for 24 h (A) and 48 h (B), and 616 in Caco-2 cells exposed to the same mixture for 24 h (C) and 48 h (D). Each point 617 represents the CI ± s.d. at a fractional effect. The dotted line (CI = 1) indicates 618 additivity, the area under the dotted line synergy, and the area above the dotted line 619 antagonism. 620 23 Figure 6. Morphology of HepG2 cells exposed to 31.91, 15.95, and 7.98 µg/ml of the 621 extract ST-99 after 24 h. Control HepG2 cells in normal growth with normal morphology 622 showing big euchromatic nuclei (N) with compact nucleoli (n) (A). Cell treated with 7.98 623 µg/ml of ST-99 developed cisternae from rough endoplasmic reticulum (rer) linked to 624 mitochondrial organelles (m) (B). Cellular interactions (arrow head) with microvilli 625 (arrow) are also observed (C). Cells showed cellular interactions (arrow) (D) and 626 apoptotic nuclei (ApN) (E). Cells exposed to 31.91 µg/ml showed cytoplasmatic 627 projections that would turn into apoptotic bodies (arrow) (F). Big lipid drops are also 628 shown (Lip) (G). Increase in the number of apoptotic cells (ApN) (H). 629 Figure 7. Morphology of HepG2 cells exposed to 29.47 (A, B, C) and 7.37 µg/ml of the 630 mixture of stilbenes (D, E, F). HepG2 cells exposed to 7.37 µg/ml of the mixture of 631 stilbenes showed cytoplasmic evaginations (arrow) (A, B) and apoptotic nuclei (ApN) 632 (C). HepG2 treated with 29.47 µg/ml of the stilbenes mixture also showed apoptotic 633 nuclei (ApN) and lipid drops (Lip) (D). At this concentration, the nucleoli (n) was in 634 segregation process of their fibrillar (f) and granular (g) components (E). However, cell 635 proliferation is still observed in mitotic process (Mit) (F). 636 637 24 Table legend 638 Table 1. Cytotoxicity of the stilbenes extract, trans-ε-viniferin, trans-resveratrol and its 639 mixture on the selected biomarkers according to EC50 values (µg/ml). 640 Table 2. The parameter m, Dm and r are the antilog of x-intercept, the slope and the 641 linear correlation coefficient of the median-effect plot, which signifies the shape of the 642 dose-effect curve, the potency (IC50), and the conformity of the data to the mass-action 643 law, respectively. 644 Tested compounds EC50 HepG2 (µg/mL) EC50 Caco-2 (µg/mL) Time of exposure ST-99 extract 31.91 ± 1.55 27.79 ± 2.35 24h 26.58 ± 2.00 19.29 ± 1.02 48h Mixture 29.47 ± 3.54 74.34 ± 2.40 24h 26.57 ± 1.92 38.67 ± 2.02 48h Trans-Ɛ-viniferin 28.28 ± 2.15 36.72 ± 3.01 24h 17.85 ± 3.03 20.63 ± 1.25 48h Trans-resveratrol >50 >50 24h 39.56 ± 2.41 48.89 ± 2.99 48h Stilbene Time Dm (µg/mL) m r Time Dm (µg/mL) m r trans-resveratrol 24h 48h 49.09 58.49 0.98 2.00 0.98 0.99 24h 48h 64.70 90.19 1.90 1.38 0.99 0.96 trans-Ɛ-viniferin 24h 48h 39.51 17.84 1.60 1.44 0.96 1.00 24h 48h 39.29 23.30 1.10 1.39 0.99 0.99 Mixture 24h 48h 59.72 60.80 1.00 1.22 0.99 0.97 24h 48h 61.99 67.35 1.26 1.20 0.99 0.99 HepG2 Caco-2 HepG2 Caco-2